TD HEWITSON1,2, SG HOLT1,2, SJ TAN1, B WIGG1, CS SAMUEL3, ER SMITH1,2
1Department of Nephrology, The Royal Melbourne Hospital, Parkville, Melbourne, VIC; 2Department of Medicine-RMH, University of Melbourne, Parkville, Melbourne, VIC; 3Department of Pharmacology, Monash University, Clayton, Melbourne, VIC, Australia.
Aim: To examine the distribution and acquisition of epigenetic modifications to histone 3 Lysine 9 (H3K9) after injury and stimulation with the pro-fibrotic cytokine TGF-b1.
Background: Epigenetic regulation of fibrogenesis through post translational histone modifications (marks) may be a key determinant of progression in renal disease.
Methods: Immunofluorescent microscopy was used to examine global H3K9 acetylation (H3K9Ac) and tri-methylation (H3K9Me3) after unilateral ureteric obstruction (UUO) in mice. Confocal, super-resolution microscopy and flow cytometry were used to determine the in vitro effect of TGF-b1 on structural arrangement of these marks, and their relationship with a- smooth muscle actin (aSMA) expression, a marker of myofibroblasts and early epithelial mesenchymal transition (EMT).
Results: The number of individual histone marks was increased 10 days after UUO (p<0.05 vs control), with both clearly seen in various cell types. Sub-nuclear microscopy in primary rat renal fibroblasts and a proximal tubule cell line (NRK-52e) showed that H3K9Ac was co-localised with phosphorylated-Ser2 RNA polymerase II (pRNAPol II), while H3K9Me3 was not, consistent with permissive and repressive effects on gene expression respectively. In both cell types H3K9Ac was diffusely distributed throughout the nucleus, while H3K9Me3 was found in compartments resembling the nucleolus, and in the case of the fibroblast, also juxtapositioned with the nuclear membrane. TGF-b1 had no effect on H3K9Ac marks in either cell, but resulted in a redistribution of H3K9Me3 within the fibroblast nucleus. This was unrelated to mitogenesis, but was associated with increased aSMA expression.
Conclusions: These findings highlight why it is important to consider the epigenetics of each cell individually. Whilst no overall enrichment occurred, renal myofibroblast differentiation was accompanied by distinct changes in histone mark arrangements.